Uses of Parylene Membrane Filters

ABSTRACT

The invention provides parylene membrane filters, filter devices and methods of making them and using them in the mechanical separation of cells and particles by size. The provision of parylene membrane filters with high figures of merit and finely controlled hole sizes allows the separation of cells and particles in a variety of biological and other fluids according to sizes.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. Nos.11/408,499, filed Apr. 20, 2006, and 11/408,501, filed Apr. 20, 2006,which both claim priority to U.S. Provisional Patent Application No.60/673,571, filed Apr. 21, 2005, which are hereby incorporated byreference in its entirety for all purposes.

FIELD OF THE INVENTION

This invention relates to polymeric membranes useful in the mechanicalfiltration of particles and cells in a fluid.

BACKGROUND OF THE INVENTION

A mechanical filter can be used to remove, filter, collect, concentrateand analyze particles and cells in a variety of fluid medium.

The filtration of cells can aid in the diagnosis of disease. Forinstance, one of the most important determinants of prognosis andmanagement of cancer is the absence or presence of metastaticdissemination of tumor cells at the time of initial presentation andduring treatment. The early spread of tumor cells to lymph nodes or bonemarrow is referred to as circulating tumor cells (CTC) when in theperipheral blood. It has been well established that these CTC can bepresent even in patients who have undergone complete removal of theprimary tumor. The detection of CTC has proven to be a useful tool indetermining the likelihood of disease progression. Similarly, theability to detect, collect, or obtain other kinds of cells (e.g.,bacterial cells, immune system cells, fetal cells) found in biologicalfluids by filtration methods will be of great clinical value both indiagnostics and therapeutics.

Further, with respect to blood and CTC, the predominance of the redblood cell can make it difficult to detect or obtain cells found inblood at much lower levels. CTC exist in blood on the order of 1 per 10billion blood cells. Currently available technologies are inadequate toidentify circulating tumor cells with the requisite sensitivity,efficiency and specificity. Existing technologies using magnetic beads,density-gradient centrifugation or polycarbonate filtration for thecapturing of tumor cells typically have poor recovery rates and extendedprocessing time in the order of hours.

One way of separating cells is by size. Many research groups havereported cell separation using mechanical filtering. For example, it wasdemonstrated a weir-type filter with a 3.5μ gap could isolate leukocyteswith a 7% capturing efficiency and >99% erythrocyte rejection (see,Wilding P, et al., Anal Biochem. 257(2):95-100 (1988); Yuen P K, et al.,Genome Res. 11(3):405-12 (2001); and Mohamed H, et al., IEEE TransNanobioscience 3(4):251-6 (2004). This work indicated an attractiveadvantage of such devices in obviatin the need to formulate a specialbuffer condition for separation. More work was, however, warranted toimprove the capturing efficiency.

The mechanical filtration method can be applied to circulating tumorcells in blood as these blood cells can substantially differ in sizefrom other major cell types found in blood:

Variability of cell sizes across different cultured tumor cells PBL:peripheral blood lymphocytes (see, Vona G, et al., Am J Pathol. 156(1):57-63 (2000) Approximate size Cell Culture Tumor Type in μ MDA-468Breast NA MCF-7 Breast 21 ± 2* J82 Bladder 16 ± 2  T24 Bladder NA PC3Prostate NA LNCaP Prostate 23 ± 2* Peripheral Blood 12 ± 2* Lymphocytes

To handle large sample volumes, as may be required when only a fewcancer cells of interest my exist in a large blood volume, a membranefilter needs a large throughput capability. Several groups havedemonstrated separation of tumor cells from whole blood withcommercially available polycarbonate filters (see, Vona G, et al., Am JPathol. 160(1):51-8 (2002); Kahn H J, et al., Breast Cancer Res Treat;86(3):237-47 (2004)). However, the existing commercial membrane filterswhich have been used to mechanically filter blood have low recoveryrates. Such filters frequently contain randomly and sparsely distributedholes, with many of them noticeably fused, resulting in large openingsthat can contribute to lower recovery rates.

Micromachined membrane filters, which have precise geometrical andthickness control, can have better performance. The opening factor canbe large and without the defects of fused doublet or triplet holes.Previously, we have demonstrated the manufacture and use of a siliconnitride/parylene membrane filter to separate particles. The particlemembrane filters (8×8 mm²) have circular, hexagonal or rectangularthrough holes. By varying hole dimensions from 6 to 12 μm, openingfactors from 4 to 45% were achieved (see, U.S. Patent ApplicationPublication No. US2001/0019029).

Therefore, there is a need for an improved filtration system which canbe used to remove, isolate, capture or detect a cell or other particlein blood or other body fluids. This invention provides for these andother needs.

BRIEF SUMMARY OF THE INVENTION

In a first aspect, the present invention provides novel membrane filterscomprising a parylene substrate and filter devices housing the parylenemembrane filter. Advantageously, the membrane filters and devices canprovide a highly efficient means for capturing or trapping particles andcells according to their size and shape. For instance, lowconcentrations of circulating tumor cells (CTC) in blood can be trappedin a parylene membrane filter having suitably sized holes which bar thepassage of a CTC while allowing red blood cells to pass through. In asecond aspect, the invention provides methods of using the parylenemembrane filters and filter devices to capture or isolate particles andcells.

Accordingly, in the first aspect, the invention provides a parylenemembrane filter. The parylene membrane of the filter is made of asubstrate comprising a parylene polymer. In a preferred embodiment, theparylene membrane is deposited through a highly-conformal vapordeposition process. Suitable types of parylene include parylene C, F, A,AM, N, and D, as well as those further described herein.

The parylene membrane filter comprises a plurality of holes of apredetermined geometric design formed in, and penetrating, the thicknessof the parylene membrane. The geometric design includes, for example, asize, a shape and density. In one embodiment, the design of the membraneis such that CTC are selectively captured or retained by the membranewhile other cells and materials in the blood pass through the membraneselected according to their size and shape. The efficiency of themembrane filter can be optimized by adjusting the size, shape anddensity of the holes on the membrane. The predetermined geometric designis according to any one or more of the size, shape, density, uniformity,and arrangement of the holes in the parylene membrane. In someembodiments of the above, the parylene membrane filter is from 0.5 to 20microns thick.

The invention also provides membrane filter devices. These devicescomprise a parylene membrane filter as described above and a housing inwhich the parylene membrane filter is mounted. In some embodiments, thedevice comprises a first chamber and a second chamber separated by theparylene membrane filter. In some embodiments, the parylene membranefilter substrate is in contact with a metal layer on an externalsurface. In some embodiments, the membrane filter device has a pluralityof parylene membrane filters having an array of holes which may be ofthe same or different predetermined geometric design.

According to a second aspect of the present invention, a method offorming a parylene membrane filter and a parylene membrane filter deviceis provided. The method typically includes the formation of a parylenemembrane having an array of holes with a predetermined geometric designand the assembly of the membrane in a housing. The predeterminedgeometric design includes the precisely controlled size, shape anddensity of the holes. In one embodiment, the geometric design includesan array of monodispersed holes.

In a third aspect, the invention is drawn to methods of using theparylene membrane filters according to the invention to isolateparticles or cells. In this aspect, the invention provides a method forisolating a particle or cell by obtaining a sample containing theparticle or cell and passing the sample through a parylene membranefilter. The size and shape of the holes can be determined empirically,for example, by determining the ability of the holes to control thepassage through the membrane of the particle or cell or of particles orcells of similar size and shape to the cell to be isolated.

In any of the above embodiments, the isolated cell may be further usedor manipulated. For instance, the isolated cells may be detected,counted, cultured, characterized, concentrated, or obtained for furtheruse (e.g., administration in a cell-based therapy).

In further embodiments, the invention provides methods for monitoringthe health status of a patient having a disease caused by a harmful cellby obtaining a sample of a body fluid containing the harmful cell andisolating the harmful cell by passing the sample through a membranefilter having a parylene substrate having a plurality of holes of apredetermined geometric design; and detecting the isolated harmful cell.

In some embodiments, the invention provides methods for monitoring thehealth status of a patient having a disease caused by a deficiency of abeneficial cell by obtaining a sample of a body fluid containing thebeneficial cell and isolating the beneficial cell by passing the samplethrough a membrane filter having a parylene substrate having a pluralityof holes of a predetermined geometric design; and detecting the isolatedbeneficial cell.

In another set of embodiments, the invention provides methods forevaluating the therapeutic efficacy of a treatment for a conditioncaused by a harmful cell, the method comprising obtaining a sample of abody fluid containing the harmful cell during or after the treatment;and isolating the harmful cell by passing the sample through a membranefilter having a parylene substrate fluid having a plurality of holes ofa predetermined geometric design; and detecting the retained harmfulcell in the sample.

In some embodiments, an insoluble particle is isolated from a sample.The sample may be obtained from a living organism, or from theenvironment (air, water, soil), or an article of human manufacture. Theparticle may be asbestos, uric acid crystals, a crystal, or an amorphoussolid. The isolated particle may then be counted or characterized.

In some embodiments, the invention provides a method of removingparticulate contaminants from a fluid by passing the sample through amembrane filter comprising a parylene substrate according to theinvention. The fluid may be a culture medium, or a drinking water, amedicine or substance for administration to a human by any route ofadministration (e.g., oral, intravenous, inhalation).

Reference to the remaining portions of the specification, including thedrawings and claims, will realize other features and advantages of thepresent invention. Further features and advantages of the presentinvention, as well as the structure and operation of various embodimentsof the present invention, are described in detail below with respect tothe accompanying drawings. In the drawings, like reference numbersindicate identical or functionally similar elements.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a fabricated membrane filter device. FIG. 1A shows aparylene film with 50 devices. FIGS. 1B and 1C show portions of one ofthe 50 devices.

FIG. 2 illustrates a typical parylene membrane filter device assembly.

FIG. 3 illustrates the configuration of a filter device assembly withmultiple parylene membranes.

FIG. 4 illustrates images of scanning electron microscope (SEM) of acommercial membrane filter and the membrane filters of the presentinvention. FIG. 4A shows an SEM image of a commercial membrane filter;FIG. 4B shows the image of a microfabricated parylene membrane filter;FIGS. 4C and 4D show images of parylene membrane filters with cellscaptured.

FIG. 5 shows hematoxylin stained tumor cells captured on the surface ofa parylene membrane filter.

FIG. 6 presents a proposed design of integrated on-chip parylenemembrane filter for tumor cell capture and detection.

DETAILED DESCRIPTION

It is noted here that as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referenceunless the context clearly dictates otherwise.

This invention provides novel parylene membrane filters and devices,including microdevices. It further provides methods of their use in thecapture, isolation, detection, and/or characterization of cells and/orparticles. Accordingly, in some embodiments, the invention providesparylene membrane filtration devices, which comprise the parylenemembrane filter and a housing. Advantageously, the parylene membranefilter allows the filtration of many fluids, including, biologicalfluids.

The parylene membrane filters and devices can provide a highly efficientmeans for capturing or trapping particles and cells in fluids accordingto their size and shape. For instance, low concentrations of circulatingtumor cells (CTC) in blood can be trapped in a parylene membrane filterhaving suitably sized holes which bar the passage of a CTC whileallowing red blood cells to pass through.

As described below, the parylene membrane filters and devices havealready demonstrated greater than 80% recovery with high enrichmentfactor, which out-performs most current methods used in the field.Moreover, less than 10 minutes can be required for each CTC captureoperation, compared to current multi-step processing requiring more thanan hour. The invention has been advantageously demonstrated as anenrichment device or cell capture device by using a model system usingcultured cancer cells admixed in blood. The novel parylene membranefilters and devices can provide a cost effective method for CTCmonitoring with higher recovery rate, faster processing and morereliable results due to minimal human intervention.

The parylene membrane filter is made of a substrate which comprises,consists essentially of, or consists of parylene as defined furtherbelow. Suitable parylenes include USP Class VI biocompatible polymers.In a preferred embodiment, the parylene membrane is deposited through ahighly-conformal vapor deposition process. Suitable types of parylenespecifically include parylene C, F, A, AM, N, and D, as well as thosefurther described herein.

The parylene membrane filter comprises a plurality of holes of apredetermined geometric design formed in, and penetrating, the parylenemembrane. The geometric design includes, for example, a size, a shapeand density. In one embodiment, the design of the membrane is such thatCTC are selectively captured or retained by the membrane while othercells and materials in the blood pass through the membrane selectedaccording to their size and shape. The efficiency of the membrane filtercan be adjusted by changing the size, shape and density of the holes onthe membrane. In some preferred embodiments, the filter of the presentinvention has a figure of merit up to 890. In other embodiments, theparylene membrane filter has a figure of merit between about 800 toabout 890. In preferred embodiments, the holes are monodispersed.

The predetermined geometric design is according to any one or more ofthe size, shape, density, uniformity, and arrangement of the holes inthe parylene membrane. In some embodiments, the holes themselves canhave rounded or sharp corners. The holes can be of a regular shape(e.g., circles, ovals, ellipses, squares, rectangles, symmetrical andunsymmetrical polygons, rods) or any other shape desired, including butnot limited to, other irregular shapes. The holes can be of differentsizes and shapes. The holes can all be of uniform size and/or shape. Inpreferred embodiments, the holes may be limited to a predetermined rangeof sizes and/or shapes. In some embodiments, membrane filter has a holeshape selected from the group consisting of a circular, an elliptical, asymmetrical polygonal, an unsymmetrical polygonal, an irregular shapeand combinations thereof. In some embodiments, the holes can be arrangedin a uniform grid or array (e.g., one or more rows and/or columns,concentric circles, and the like). Preferably, holes are all of the sameshape and size and may also be of uniform density or pattern on themembrane, aside from the edges. The size and shape of the holes can bedetermined empirically, for example, by determining the ability of theholes to control the passage through the membrane of the particle orcell or of particles or cells of similar size and shape to the cell tobe isolated.

Accordingly, the holes may be of any size and shape which will determinethe ability of a particle or cell of interest to pass through. Forinstance, in some embodiments, the holes may have a minimum or maximumcross sectional length of 1, 2, 3, 4, 5, 8, 10, 12, 14, 16, 18, 20, 24,28, 30, 32, 36, 40, 45, or 50 microns. In some embodiments, the holesare circles, ovals, or polygons. In some further embodiments, thecircular holes have diameters of 2, 4, 8, 10, 14, 20, 30, 40 or 50microns. In other further embodiments, the holes are oval and havedifferent lengths and widths which may be independently be selected from2, 4, 6, 8, 10, 14, 20, 30, 40 or 50 microns. For instance, in somefurther embodiments, the holes may be circles from 6 to 10, 5 to 12, 10to 20, 8 to 40, or 6 to 60 microns in diameter. In other embodiments,the holes may be ovals whose dimensions are from 4 to 10 microns by 11by 20 microns, from 8 to 20 microns by 8 to 20 microns, by 8 to 20microns by 8 to 40 microns, or by 8 to 80 microns by 8 to 80 microns. Insome embodiments of any of the above, the minimum width of hole is 2, 4,6, 8, or 10 microns. In some embodiments, of any of the above themaximum length of the hole is 50, 100 microns or 200 microns.

The holes may also be defined according to their cross sectional areaand/or shape. The shapes can be as any described above. In someembodiments, the cross sectional areas range from about 1 to 1000 squaremicrons, 1 to 10 square microns, 10 to 100 square microns, 25 to 500square microns, 50 to 400 square microns, 75 to 150 square microns, 75to about 500 square microns, or 200 to 1000 square microns. In any ofthe above, the holes may be monodispersed. In any of the above, theparylene membrane filter may have a figure of merit up to 890, andpreferably from 800 to 890.

In some embodiments, the parylene membrane filter has a hole density offrom 5,000 to 40,000; 10,000 to 40,000; 10,000 to 30,000; 20,000 to30,000; 20,000 to 40,000; or 30,000 to 40,000 holes per squaremillimeter. Such hole densities depend in part upon the sizes of theholes, with smaller holes allowing for greater densities. The densitiescan be adjusted so as to insure that the holes do not fuse togetherduring manufacture and the strength of the parylene membrane remainssuitable. A thicker membrane can be used to strengthen the membrane athigher hole densities.

In certain instances, the number and size of the holes affects the rateat which a sample can pass through the membrane and the strength of themembrane. The density of the holes will typically range from 1,000 to40,000 holes per square millimeter. The plurality of holes can providean opening area ratio of from 4% to 60%, including ranges from 4% to25%, 5% to 25%, 10% to 25%, 15% to 30%, 5% to 45%, 10% to 50%, 15% to45%, 20% to 40%, 25% to 50%, and 45% to 60%.

In some embodiments of the above, the parylene membrane filter is from0.5 to 20 microns thick. In preferred embodiments, the membrane is atleast 1 micron thick. In other embodiments, the membrane filter is from1 to 10 microns thick, in more preferred embodiments, the membranefilter is from 1 to 4 microns thick. The thickness of the membranefilter is a compromise between membrane strength and flow resistancethrough the membrane. Accordingly, as increasing hole density reducesmembrane strength, membranes having a greater number of holes typicallyrequire a thicker membrane than membranes having a fewer number of thesame holes.

In some embodiments of the above, the parylene membrane filter itself ormembrane filter surface area is provided in various sizes and shapes.The membrane filter or each filtering surface of the membrane can be inthe form of a circle, oval, symmetrical or asymmetrical polygon, squarerectangle, or an irregular shape. The membrane filter or each filteringsurface of the membrane may have a surface area from 4 squaremillimeters to 10 square centimenter, from 10 square millimeters to 100square millimeters, from 25 square millimeters to 625 squaremillimeters, or from 25 square millimeters to 250 square millimeters, orfrom 25 square millimeters to 100 square millimeters. The cross sectionof the membrane filter or filter surface portion thereof in part isgoverned or in accordance with the amount of fluid to be filtered, theconcentration of the retained particles or cells in the fluid, thepressure to be applied, and the strength of thickness of the membrane.In some instances, the membrane filters may be mounted separately in ahousing and in parallel.

The invention also provides membrane filter devices. These devicescomprise a parylene membrane filter as described above and a housing inwhich the parylene membrane filter is mounted. In some embodiments, thedevice comprises a first chamber and a second chamber separated by theparylene membrane filter. In some embodiments, the parylene membranefilter substrate is in contact with an external metal layer. In somefurther embodiments, the metal is selected from the group consisting ofAu, Pt, Ag, Pd, Cu, Ir, Zn, Ni, Fe, Ru, Rh and Si. In some embodiments,the metal does not comprise Si.

In some embodiments, the membrane filter device has a plurality ofparylene membrane filters having an array of holes which may be of thesame or different predetermined geometric design. For example, the firstmembrane filter may have a circular hole and the second membrane filteran oval hole. In some embodiments, the plurality of parylene membranefilters are disposed substantially in parallel. The device may have oneor more stacks of multiple membrane filters. In some preferredembodiments, the plurality of parylene membrane filters are disposedsequentially in a housing. For instance, the filter device may comprisefirst parylene membrane filter having an array of holes with a firstpredetermined geometric design; and a second parylene membrane filterhaving an array of holes with a second predetermined geometric design,wherein the first membrane filter is disposed above said second membranefilter. In some embodiments, the device may further comprise a sandwichlayer disposed between said first membrane filter and said secondmembrane filter. The sandwich layer may be an inert layer. In someembodiments, the sandwich layer does not comprise Si.

According to a second aspect of the present invention, a method forforming a parylene membrane filter and a parylene membrane filter deviceis provided. The method typically includes the formation of an array ofholes with a predetermined geometric design in an area of a parylenemembrane and the assembly of the membrane in a housing. Thepredetermined geometric design includes the precisely controlled size,shape and density of the holes. In one embodiment, the geometric designincludes an array of monodispersed holes.

This fabrication process can be used for various membrane filters. Thepreferred hole shapes include circular, hexagonal, and/or rectangular.Filters as large as 8 by 8 square millimeters can be fabricated. Otherfactors being constant, the opening area ratio increases as the holesize increases. The hole size also defines the filtering threshold—theminimum size of the particles that can be blocked by the filter.

For example, a filter with a 10.6 micron diameter hole has an openingarea ratio of approximately 12½%. Hexagonal holes can provide higheropening area ratios, but provide corners which cause higher stressconcentration in the membrane. This effectively reduces the strength ofthe filter. Rectangular holes can provide a large range of opening arearatio without changing the filtering threshold. In some embodiments, onedimension of the rectangular holes must be kept constant.

It is now possible to fabricate complex miniaturized systems or“microdevices.” This technology represents a combination of severaldisciplines that include microfabrication, microfluidics,microelectronmechanical systems, chemistry, biology, and engineering.Miniaturized devices can be electrical, such as microelectrodes andsignal transducers; optical such as photodiodes and optical waveguides;and mechanical, such as pumps. In the new field of microfluidics, theintegration of automated microflow devices and sensors allow veryprecise control of ultra-small flows on microchip platforms (Gravesen etal. (1993) J. Micromech. Microeng. 3:168-182; Shoji and Esashi (1994) J.Micromech. Microeng. 4:157-171). Many different flows can be combined inall sorts of ways and mixed on the same chip. Existing technology alsoallows the integration of intersecting channels, reaction chambers,mixers, filters, heaters, and detectors to perforin on-chip reactions insub-nanoliter volumes in a highly controlled and automated manner withintegrated data collection and analysis (Colyer et al. (1997)Electrophoresis 18:1733-1741; Effenhauser et al. (1997) Electrophoresis12:2203-2213).

In some embodiments, the parylene membrane filters can be integrated aspart of a “microdevice” for filtering a sample. The term “microdevice”is used to describe miniaturized sensing devices and systems thatintegrate microscopic versions of the devices necessary to processchemical or biochemical samples, thereby achieving completely automatedand computer controlled analysis on a microscale (typically handling 100microliter sample volumes or less). Microdevices may be classified intotwo groups. One is a MEMS (Micro Electro Mechanical System), which usespressurized flow controlled by mechanical flow control devices (e.g.,microvalves, micropumps or centrifugal pumps). The other types useelectrically driven liquid handling without mechanical elements. Avariety of integrated these devices are well known to the art. See, forexample, U.S. Pat. Nos. 6,043,080; 6,042,710; 6,042,709; 6,036,927;6,037,955; 6,033,544; 6,033,546; 6,016,686; 6,012,902; 6,011,252;6,010,608; 6,010,607; 6,008,893; 6,007,775; 6,007,690; 6,004,515;6,001,231; 6,001,229; 5,992,820; 5,989,835; 5,989,402; 5,976,336;5,972,710; 5,972,187; 5,971,355; 5,968,745; 5,965,237; 5,965,001;5,964,997; 5,964,995; 5,962,081; 5,958,344; 5,958,202; 5,948,684;5,942,443; 5,939,291; 5,933,233; 5,921,687; 5,900,130; 5,887,009;5,876,187; 5,876,675; 5,863,502; 5,858,804; 5,846,708; 5,846,396;5,843,767; 5,750,015; 5,770,370; 5,744,366; 5,716,852; 5,705,018,5,653,939; 5,644,395; 5,605,662; 5,603,351; 5,585,069; 5,571,680;5,410,030; 5,376,252; 5,338,427; 5,325,170; 5,296,114; 5,274,240;5,250,263; 5,180,480; 5,141,621; 5,132,012; 5,126,022; 5,122,248;5,112,460; 5,110,431; 5,096,554; 5,092,973; 5,073,239; 4,909,919;4,908,112; 4,680,201; 4,675,300; and 4,390,403, all of which areincorporated by reference herein.

In a third aspect, the invention is drawn to methods of using theparylene membrane filters according to the invention to isolateparticles or cells. In this aspect, the invention provides a method forisolating a particle or cell by obtaining a sample containing theparticle or cell and passing the sample through a parylene membranefilter. The size and shape of the holes can be determined empirically,for example, by determining the ability of the holes to control thepassage through the membrane of the particle or cell or of particles orcells of similar size and shape to the cell to be isolated.

In some embodiments, the sample is passed through a succession ofmembrane filters wherein each membrane filter comprises a parylenemembrane having a plurality of holes wherein the holes of eachsucceeding parylene membrane are of smaller cross-sectional area thanthose of the parylene membrane of the preceding membrane filter, and thecell or particle is retained on one of the succession of membranefilters. In some embodiments, after the sample passes through the firstmembrane filter the cell or particle is retained on the upstream surfaceof a successive membrane filter. Two, three, four, five or more suchmembranes may be arranged in succession. In some embodiments, asuccessive membrane filter comprises a parylene membrane having aplurality of holes of a predetermined geometric design which differsfrom that of preceding membrane with regard to shape, density,arrangement, or opening area ratio.

For instance, the sample can be passed through a second membrane filter,wherein the second membrane filter comprises a second parylene membranehaving a second plurality of holes of a second predetermined geometricdesign, wherein each of the plurality of holes of the second parylenemembrane have a cross-sectional area which is smaller than thecross-sectional area of each of the holes of the first parylenemembrane, and the cell or particle passes through the first membranefilter and is retained on the surface of the second membrane filter.

In some embodiments, a cell is to be isolated. In these embodiments, asample is obtained which contains the cell and the sample is passedthrough a membrane filter which retains the cell but allows othercomponents of the sample to pass through. For instance, the membranefilter may comprise a parylene substrate having a first plurality ofholes of a predetermined geometric design which prevent the cell frompassing through the membrane while allowing other smaller particles orsoluble components to pass through. For instance, the cell may be of asize and shape which is too large to pass through the holes. The cellmay be retained by the membrane filter when it has a minimumcross-sectional area greater which is greater than a maximalcross-sectional are of the predetermined geometric design. In someembodiments, the smallest achievable cross section of a deformable cellmay be larger than the largest cross-section of each of the holes of thefirst predetermined design.

In other embodiments, where a cell is to be isolated, a samplecontaining the cell is obtained and passed through a membrane filteraccording to the invention which retains other components of the sample,but not the cell. For instance, the membrane filter may comprise aparylene substrate having a first plurality of holes of a predeterminedgeometric design which allow the cell to passing through the holes. Forinstance, the cell may be of a size and shape which is small enough topass through the holes or too small to be retained by the membranefilter. The cell may pass through the membrane when it has a minimumcross-sectional area greater which is smaller than a maximalcross-sectional are of the predetermined geometric design.

In some embodiments, the sample can contain a cell of a first type and acell of a second type and the predetermined geometric design selectivelyallows the cells of the second type to pass through the membrane filterwhile selectively limiting the passage of the cells of the first type.

The sample can be a body fluid or any fluid containing a cell. Thesample can, for instance, urine, cerebrospinal fluid, saliva, peritonealfluid, cardiac fluid, pericardial fluid, pleural fluid, blood, orplasma, semen, fluid from a wound or infection, bodily discharge, ormucous. The sample can be a disaggregated portion of a tissue, includingdisaggregated portions of a solid tumor. The cell to be isolated can beof any cell type, including, but not limited to, bacteria, yeast,eukaryotes, prokaryotes, and mammalian cells. In a preferred embodiment,the cell is tumor cell found in a body fluid. For instance, the cell canbe a circulating tumor cell found in blood. The circulating tumor cellmay further be from a solid tumor or of a solid-tumor cell type.

In some embodiments, the sample is from a mammal (e.g., primates,humans, mice, rats, rabbits). In further such embodiments, the cell canbe a cancer cell, fetal cell, stem cell (e.g., cardiac stem cell, liverstem cell, neuronal stem cell, hematopoietic stem cells, endothelialstem cell). In some such embodiments, the sample is blood or a bloodproduct bone marrow (e.g., aspirated, disaggregated bone marrow) or cordblood.

The mammalian cell can be of any cell, tissue type, or organ. It can bean epithelial cell, endothelial cell, nerve, immune system, atransformed cell, or a cell from the lung, liver, blood, liver, kidney,muscle, heart, brain, prostate, breast, bladder, esophagus, stomach,colon, mucosa, gastrointestinal tract. The cell can be a red blood cell,white blood cell, monocyte, dendritic cell, T-cell, B-cell. In someembodiments, the cell is a tumor cell or a circulating tumor cell of anytissue of origin, including those described above. The cell can be aleukemic cell or a lymphoma or a cancer cell.

“Cancer” refers to human cancers and carcinomas, sarcomas,adenocarcinomas, lymphomas, leukemias, and the like, including solidtumors and lymphoid cancers, kidney, breast, lung, kidney, bladder,colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin,uterine, testicular, esophagus, and liver cancer, lymphoma, includingnon-Hodgkins and Hodgkins lymphoma, leukemia, and multiple myeloma.“Urogenital cancer” refers to human cancers of urinary tract and genitaltissues, including but not limited to kidney, bladder, urinary tract,urethra, prostrate, penis, testicle, vulva, vagina, cervical and ovarytissues.

In some samples, the cell is a bacteria. In some further embodiments ofsuch, the sample is urine, blood, saliva, saliva, a mucosal fluid,cerebrospinal fluid, an exudate from a would or infection, or a bodilyfluid.

The sample can be fresh or salt water and the cell a single ormulticelled organism (e.g., plankton, bacteria, fungi, protozoa, algae,amoeba, paramecium, protists). In some such embodiments, the inventionprovides a method of removing harmful organisms from drinking water.

A “sample” is a medium containing a substance of interest, synthetic ornatural, to be examined, treated, determined or otherwise processed todetermine the amount or effect of a known or unknown analyte therein.The sample to be filtered can be a “biological sample.” A biologicalsample includes sections of tissues such as biopsy and autopsy sampleswhich are disaggregated prior to filtration and suspension in a fluidmedium. Such samples include blood and blood fractions or products(e.g., serum, plasma, platelets, red blood cells, and the like), sputum,tissue, cultured cells, e.g., primary cultures, explants, andtransformed cells, stool, urine, etc. A biological sample is typicallyobtained from a eukaryotic organism, most preferably a mammal such as aprimate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guineapig, rat, Mouse; rabbit; or a bird; reptile; or fish.

A sample may be filtered directly, if liquid, or suspended or diluted ina suitable liquid medium. Preferred medium for cells are physiologicallycompatible aqueous fluids and buffers (e.g., phosphobuffered saline,growth medium if a viable cell is to be isolated) which are free ofextraneous particles. Preferred medium for particles are those which arefree of extraneous particles and do not dissolve the particle ofinterest and which are compatible with the parylene membrane filter. Aparticularly preferred medium for most water-insoluble particles isaqueous. The medium may include components which enable detection of theparticle or cell or reduce viscosity.

A “biopsy” refers to the process of removing a tissue sample fordiagnostic or prognostic evaluation, and to the tissue specimen itself.Any biopsy technique known in the art can be applied to the diagnosticand prognostic methods of the present invention. The biopsy techniqueapplied will depend on the tissue type to be evaluated (i.e., prostate,lymph node, liver, bone marrow, blood cell), the size and type of thetumor (i.e., solid or suspended (i.e., blood or ascites)), among otherfactors. Representative biopsy techniques include excisional biopsy,incisional biopsy, needle biopsy, surgical biopsy, and bone marrowbiopsy. An “excisional biopsy” refers to the removal of an entire tumormass with a small margin of normal tissue surrounding it. An “incisionalbiopsy” refers to the removal of a wedge of tissue that includes across-sectional diameter of the tumor. A diagnosis or prognosis made byendoscopy or fluoroscopy can require a “core-needle biopsy” of the tumormass, or a “fine-needle aspiration biopsy” which generally obtains asuspension of cells from within the tumor mass. Biopsy techniques arediscussed, for example, in Harrison's Principles of Internal Medicine,Kasper, et al., eds., 16th ed., 2005, Chapter 70, and throughout Part V.The samples may be enzymatically disaggregated prior to filtration asknown to one of ordinary skill in the art.

“Antibody” refers to a polypeptide comprising a framework region from animmunoglobulin gene or fragments thereof that specifically binds andrecognizes an antigen. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon, and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes. Lightchains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.Typically, the antigen-binding region of an antibody will be mostcritical in specificity and affinity of binding.

An exemplary immunoglobulin (antibody) structural unit comprises atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms variable light chain(V_(L)) and variable heavy chain (V_(H)) refer to these light and heavychains respectively.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. Thus, for example, pepsin digests an antibody below thedisulfide linkages in the hinge region to produce F(ab)′₂, a dimer ofFab which itself is a light chain joined to V_(H)-C_(H)1 by a disulfidebond. The F(ab)′₂ may be reduced under mild conditions to break thedisulfide linkage in the hinge region, thereby converting the F(ab)′₂dimer into an Fab′ monomer. The Fab′ monomer is essentially Fab withpart of the hinge region (see Fundamental Immunology (Paul ed., 3d ed.1993). While various antibody fragments are defined in terms of thedigestion of an intact antibody, one of skill will appreciate that suchfragments may be synthesized de novo either chemically or by usingrecombinant DNA methodology. Thus, the term antibody, as used herein,also includes antibody fragments either produced by the modification ofwhole antibodies, or those synthesized de novo using recombinant DNAmethodologies (e.g., single chain Fv) or those identified using phagedisplay libraries (see, e.g., McCafferty et al., Nature 348:552-554(1990))

For preparation of antibodies, e.g., recombinant, monoclonal, orpolyclonal antibodies, many techniques known in the art can be used(see, e.g., Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al.,Immunology Today 4: 72 (1983); Cole et al., pp. 77-96 in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc. (1985); Coligan,Current Protocols in Immunology (1991); Harlow & Lane, Antibodies, ALaboratory Manual (1988); and Goding, Monoclonal Antibodies: Principlesand Practice (2d ed. 1986)). The genes encoding the heavy and lightchains of an antibody of interest can be cloned from a cell, e.g., thegenes encoding a monoclonal antibody can be cloned from a hybridoma andused to produce a recombinant monoclonal antibody. Gene librariesencoding heavy and light chains of monoclonal antibodies can also bemade from hybridoma or plasma cells. Random combinations of the heavyand light chain gene products generate a large pool of antibodies withdifferent antigenic specificity (see, e.g., Kuby, Immunology (3^(rd) ed.1997)). Techniques for the production of single chain antibodies orrecombinant antibodies (U.S. Pat. No. 4,946,778, U.S. Pat. No.4,816,567) can be adapted to produce antibodies to polypeptides of thisinvention. Also, transgenic mice, or other organisms such as othermammals, may be used to express humanized or human antibodies (see,e.g., U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;5,633,425; 5,661,016, Marks et al., Bio/Technology 10:779-783 (1992);Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13(1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996);Neuberger, Nature Biotechnology 14:826 (1996); and Lonberg & Huszar,Intern. Rev. Immunol. 13:65-93 (1995)). Alternatively, phage displaytechnology can be used to identify antibodies and heteromeric Fabfragments that specifically bind to selected antigens (see, e.g.,McCafferty et al., Nature 348:552-554 (1990); Marks et al.,Biotechnology 10:779-783 (1992)). Antibodies can also be madebispecific, i.e., able to recognize two different antigens (see, e.g.,WO 93/08829, Traunecker et al., EMBO J. 10:3655-3659 (1991); and Sureshet al., Methods in Enzymology 121:210 (1986)). Antibodies can also beheteroconjugates, e.g., two covalently joined antibodies, orimmunotoxins (see, e.g., U.S. Pat. No. 4,676,980, WO 91/00360; WO92/200373; and EP 03089).

In some embodiments, the cell to be captured is first modified to changeits effective size. In one embodiment, the cell is modified bycovalently or non-covalently attaching the cell to a particle of a sizewhich is excluded from passage through a parylene membrane filter. Inthis fashion, the cell to be captured can be isolated from cells orparticles of otherwise a similar or smaller size than the cell ofinterest as these other cells will pass through a filter when a samplewith the cell to be captured is passed through a parylene membranefilter. For instance, the cell can be contacted with an antibody orantibody fragment or another binding agent (e.g., ligand for a cellsurface receptor on the cell to be captured), which is selective orspecific with respect to the cell to be captured and which is covalentlyor non-covalently attached to the particle. In some embodiments, theparticle may be a polymeric, ceramic, glass, or metal and may be in theform of a bead or have an irregular shape. In some embodiments, theattachment may be fashioned to be readily severed by a subsequenttreatment (e.g., enzymatic, chemical, or photolytic cleavage of suitablylabile linkers; addition of competitive binding ligands which disruptthe attachment, etc.). In such embodiments, upon cleavage of theattachment, the captured cell is then able to pass through the parylenemembrane filter isolated from particles of a similar size which passedthrough the filter.

Many coupling agents are known in the art and can be used to immobilizethe antibodies and ligands and labels in the methods of the presentinvention. Coupling agents are exemplified by bifunctional crosslinkingreagents, i.e., those which contain two reactive groups which may beseparated or tethered by a spacer. These reactive ends can be of any ofa number of functionalities including, without limitation, aminoreactive ends such as N-hydroxysuccinamide, active esters, imidoesters,aldehydes, epoxides, sulfonyl halides, isocyanate, isothiocyanate,nitroaryl halides, and thiol reactive ends such as pyridyl disulfide,maleimides, thiophthalimides and active halogens.

As described in U.S. Pat. No. 4,824,529, hydroxyl functional groups arecommonly introduced to the surfaces of glasses, semiconductors, metaloxides, metals and polymers. These hydroxyl groups react withcommercially available linkers such as (3-aminopropyl) triethyloxysilaneor with thiol-terminal silanes, for example. To these amino orthiol-terminal silanes one may then graft the desired peptide, protein,lipidic, or glycosidic moiety via homobifunctional crosslinkers such asgluteraldehyde or via heterobifunctional crosslinkers.

Proteins and nucleic acids have been immobilized onto solid supports inmany ways. Methods used for immobilizing such are described in thefollowing references, and others (Mosbach (1976) Meth. Enzymol.44:2015-2030; Weetall (1975) Immobilized Enzymes, Antigens, Antibodiesand Peptides; Hermanson, G. T. (1996) Bioconjugate Techniques (AcademicPress, NY); Bickerstaff, G. (ed) (1997). Immobilization of Enzymes andCells (Humana Press, NJ); Cass and Ligler (eds) Immobilized Biomoleculesin Analysis, (Oxford University Press); Watson et al. (1990) Curr. Opin.Biotech. 609:614; Ekins, R. P. (1998) Clin. Chem. 44:2105-2030; Roda etal. (2000) Biotechniques 28:492-496; Schena et al. (1998) Trends inBiotechnol. 16:301-306; Ramsay, G. (1998) Nat. Biotechnol. 16:40-44;Sabanayagam et al. (2000) Nucl. Acids Res. 28:E33; U.S. Pat. No.5,700,637 (Southern, 1997); U.S. Pat. No. 5,736,330 (Fulton, 1998); U.S.Pat. No. 5,770,151 (Roach and Jonston, 1998); U.S. Pat. No. 5,474,796(Brenman, 1995); U.S. Pat. No. 5,667,667 (Southern, 1997); all of whichare incorporated by reference herein).

A great many cross-linkers are commercially available (e.g., from PierceChemical Company (Rockford, Ill.). A cross-linker is a molecule whichhas two reactive groups with which to covalently attach a protein,nucleic acids or other molecules. In between the reactive groups istypically a spacer group. Steric interference with the activity of thebiomolecule by the surface may be ameliorated by altering the spacercomposition or length. There are two groups of cross-linkers,homobifunctional and heterobifunctional. In the case ofheterobifunctional crosslinkers, the reactive groups have dissimilarfunctionalities of different specificities. On the other hand,homobifunctional cross linkers' reactive groups are the same. A throughreview of crosslinking can be found in Wong, 1993, Chemistry of ProteinConjugation and Cross-linking, CRC Press, Boca Raton. Bifunctionalcross-linking reagents may be classified on the basis of the following(Pierce Chemical Co. 1994): functional groups and chemical specificity,length of cross-bridge, whether the cross-linking functional groups aresimilar (homobifunctional) or different (heterobifunctional), whetherthe functional groups react chemically or photochemically, whether thereagent is cleavable, and whether the reagent can be radiolabeled ortagged with another label.

The term “label” is used herein to refer to agents or moieties that arecapable of providing a detectable signal, either directly or throughinteraction with additional members of a signal producing system. Labelsthat are directly detectable and may be used in the subject inventioninclude, for example, fluorescent labels where fluorescers of interestinclude, but are not limited to fluorescein (FITC, DTAF) (excitationmaxima, 492 nm/emission maxima, 516-525 nm); Texas Red (excitationmaxima, 595/emission maxima, 615-620); Cy-5 (excitation maxima,649/emission maxima, 670); RBITC (rhodamine-B isothiocyanate (excitationmaxima, 545-560 nm/emission maxima, 585 nm) and others as reviewed, forexample, in Haugland, R. P. (1992) Handbook of Fluorescent Probes andResearch Chemicals, 5th ed., Molecular Porbes, Eugene, Oreg.;radioactive isotopes, such as ³²S, ³²P, ³H, and the like. Other labelscan include chemiluminescent compounds, enzymes and substrates;chromogens, metals, nanoparticles, liposomes or other vesiclescontaining detectable substances. Colloidal metals and dye particlessuitable for labels are disclosed in U.S. Pat. Nos. 4,313,734 and4,373,932. Chemiluminescent and fluorescent labels allowingultrasensitive assays are preferred. Labels may be detected byspectrophotometric, radiochemical, electrochemical, chemiluminescent andother means. Labels may be covalently conjugated to binding pairmembers.

Labels may be conjugated directly to the biorecognition molecules, or toprobes that bind these molecules, using conventional methods that arewell known in the arts (see above). Multiple labeling schemes are knownin the art and permit a plurality of binding assays to be performedsimultaneously in the same reaction vesicle. Different labels may beradioactive, enzymatic, chemiluminescent, fluorescent, or others.Multiple distinguishable labels may be attached directly to biomoleculesor they may be attached to surfaces onto which the biomolecules areimmobilized. For example, beads or other particles may bear differentlabels, e.g., a combination of different fluorescent color dyes, thatallow each bead to be independently identified. For example, Fulton etal, 1997, Clin. Chem. 43: 1749-1756, describe a standard set of 64microspheres where each different type of microsphere is tagged with aunique combination of fluorescent dyes. Different biomolecules areimmobilized to each microsphere type and reacted with their binderswhich are labeled with a different color fluorescent dye. The detectorsimultaneously identifies each bead type and the captured ligand basedon the fluorescent profiles generated by the different coloredfluorescent dyes.

Preferred detectable labels include enzymatic moieties capable ofconverting a substrate into a detectable product. Enzymes are amplifyinglabels (one label leads to many signals) and facilitate the developmentof ultrasensitive assays. For example, alkaline phosphatase andhorseradish peroxidase are commonly used enzyme labels andattomole-zeptomole detection limits are routinely achieved inchemiluminescent assays with these enzymes. For alkaline phosphatase,the adamantly 1,2-dioxetane acrylphosphate substrates provideultrasensitive assays (Bronstein et al. (1989) J. Biolumin. Chemilumin.4:99-111). And for horseradish peroxidase, the 4-iodophenol-enhancedluminol reaction is among the most sensitive (Thorpe, et al, (1986)Methods Enzymol. 133:331-353). In such embodiments where an enzymaticlabel is used to convert a substrate into a detectable produce, theappropriate substrate is also added preferably after the binders havebeen captured on the surface.

Fluorescent labels are particularly useful in some embodiments of thecurrent invention. By the use of optical techniques (e.g., confocalscanners, CCD cameras, flow cytometers), they permit the analysis ofarrays of biorecognition elements distributed over a surface (e.g., asmicrodots where each microdot binds a different analyte) ordifferentially labeled (e.g., with beads having different combinationsof fluorescent dyes).

Furthermore, biotinylated binders may also be labeled in a second stepusing avidin or streptavidin (which bind biotin) conjugated to afluorophor or some other label. This labeling method is commonly used inthe art.

In any of the above embodiments, the isolated cell may be further usedor manipulated. For instance, the isolated cells may be detected,counted, cultured, characterized, concentrated, or obtained for furtheruse (administration in a cell-based therapy). The isolated cell may bedetected, counted, cultured, characterized, on the parylene membranefilter. With regard to detection and characterization, the cells may bedetected and/or characterized by contacting the cell with a histologicalstain or contacting the retained cell with a tissue-specific orcell-type specific antibody having a label. The antibody may be amonoclonal antibody. The cell may be contacted with a reagent capable ofdetecting expression of a protein (e.g., a tumor associated antigen) ora nucleic acid encoding the protein (e.g., nucleic acid encoding a tumorassociated antigen). In some embodiments, the cells are characterized asto their ability to become invasive or metastasize. In some embodiments,the cells are characterized for sensitivity or responsiveness to atherapeutic agent. In some embodiments, the cells are cultured forfurther study or autologous or heterologous transplant.

In further embodiments of the above, the invention provides methods formonitoring the health status of a patient having a disease caused by aharmful cell by obtaining a sample of a body fluid containing theharmful cell and isolating the harmful cell by passing the samplethrough a membrane filter having a parylene substrate having a pluralityof holes of a predetermined geometric design; and detecting the isolatedharmful cell. The harmful cell can be, for instance, a cancer cell or abacterium or an immune system cell. In some further embodiments, thenumber of the detected cells is counted.

In some embodiments, the isolated cell is characterized by the numberisolated or concentration in the sample and/or phenotype to determinethe likelihood of metastasis or the presences of a malignant tumor. Insome such embodiments, the sample of the body fluid is blood, or urine.Methods of characterizing the phenotype of cells with respect to theirpotential for metastasis are well known in the art.

In some embodiments of the above, the invention provides methods formonitoring the health status of a patient having a disease caused by adeficiency of a beneficial cell by obtaining a sample of a body fluidcontaining the beneficial cell and isolating the beneficial cell bypassing the sample through a membrane filter having a parylene substratehaving a plurality of holes of a predetermined geometric design; anddetecting the isolated beneficial cell. The beneficial cell can be, forinstance, an immune system cell or a stem cell. In some furtherembodiments, the number of the detected cells is counted.

In another set of embodiments, the invention provides methods forevaluating the therapeutic efficacy of a treatment for a conditioncaused by a harmful cell, the method comprising by obtaining a sample ofa body fluid containing the harmful cell during or after the treatment;and isolating the harmful cell by passing the sample through a membranefilter having a parylene substrate fluid having a plurality of holes ofa predetermined geometric design; and detecting the retained harmfulcell in the sample. In further embodiments, a sample of the body fluidcontaining the harmful cell may also be obtained before the treatment toprovide a pretreatment sample; and the harmful cell can be isolated bypassing the pretreatment sample through a membrane filter having aparylene substrate having a plurality of holes of a predeterminedgeometric design; and the isolated harmful cell in the pretreatmentsample can be compared to the isolated harmful cell in the sampleobtained during or after the treatment. The cells may be compared as tonumber, viability, or phenotype. The harmful cell may be a cancer or acell of the immune system. In some further embodiments, the treatment isselected from the group consisting of immunotherapy, chemotherapy,radiation therapy, excision of a tumor, induction of apoptosis, or anycombination thereof. In some embodiments, the therapy is an antibiotictherapy and the harmful cell is a bacteria.

In some embodiments, an insoluble particle isolated from a sample. Thesample may be obtained from a living organism or from the environment(air, water, soil) or an article of human manufacture. The particle maybe asbestos, uric acid, crystal, or amorphous solid. The isolatedparticle may then be counted or characterized.

In some embodiments, invention provides a method of removing particulatecontaminants from a fluid by passing the sample through a membranefilter comprising a parylene substrate according to the invention. Thefluid may be a culture medium, or a drinking water, a medicinal orsubstance for administration to a human by any route of administration(e.g., oral, intravenous, inhalation).

As used herein, the term “parylene” with reference to a “parylene”membrane filter refers to a polymer having formulae I, II, and III (seebelow) or combinations thereof. The polymer can be a homopolymer, acopolymer, a polymer blend or combinations thereof R¹, R², R⁷ and R⁸ areeach independently H, alkyl, heteroalkyl, aryl or halogen. The alkyl canbe a C₁-C₆ hydrocarbon radical. The halogen is Cl, F, Br, or I.Heteroalkyl is an alkyl substituent containing at least one heteroatom,such as O, S, N, Si or P.

R³-R⁶ are each independently H, alkyl, aryl, halogen, heteroalkyl,hydroxyl, amino, alkylamino, arylamino, aroylamino, carbamoylamino,aryloxy, acyl, thio, alkylthio, cyano, alkoxy. An alkyl group can be asubstituted alkyl having up to 29 carbon atoms. A substituted alkyl canbe mono- or polyunsaturated alkenyl or alkynyl radical having in eachcase up to 29 carbon atoms, i.e., a substituted C₁-C₂₉alkyl,C₂-C₂₉alkenyl or C₂-C₂₉alkynyl radical. Suitable substitutents are alsocyclic radicals. The substituted alkyls can be methyl, ethyl, or propylradical, carrying one or more identical or different radicals. Dependingon the nature of the substitutents, these can be attached via a singleor multiple bond or in a spiro form. Preferred substitutents arehalogen, such as Cl, F, Br or I, amino, lower alkylamino, loweralkanoylamino, aroylamino, such as, in particular, benzoyl amino,hyroxyamino, hydroxyimino, lower alkoxyamino, aroxyamino, such as, inparticular, phenoxyamino. Lower alkylthio includes C₁-C₆alkylthiols.Aryloxycarbonyl includes phenoxycarbonyl, benzyloxycarbonyl,hydroxyaminocarbonyl, aminoacylamino, carbamoyl, amidino. Aryoxy can bephenyloxy, aminocarbonyl-oxy, oxo, aminosulfonyl and loweralkylsulfonyl-amino. Heteroalkyl is an alkyl substitutent having one ormore heteroatoms in the alkyl substitutents, in particular,mercaptoalkyl having up to 29 carbon atoms, aminoalkyl, phosphinoalkyl,haloalkyl, hydroxyalkyl or silylalkyl. Preferably, parylene has astructure represented by the formula I. In preferred embodiments of theabove R¹, R², R⁷, and R⁸ are independently hydrogen or C₁-C₆ alkyl. Inother embodiments of the above R³ to R⁶ are independently hydrogen orC₁-C₆ alkyl. In other embodiments of the above, R¹, R², R⁷, and R⁸ areindependently hydrogen or C₁-C₆ alkyl and at least one or one of R³ toR⁶ comprises or is a functional group (e.g., amino, thio, hydroxy,halo). In some further embodiments, the halo group is chloro or fluoro.In some embodiments of any of the above, the R¹ to R⁸ members are notthemselves substituted.

Functionalized parylene polymers are also contemplated. Funtionalizedparylene includes a parylene having formula (I), wherein at least one ofthe R³ to R⁶ members is a functional group. Suitable functional groupsinclude, but are not limited to, optionally substituted amino, hydroxyl,hydroxyamino, heteroalkyl, heteroaryl, mercapto, formyl, alkanoyl,carboxylate, alkoxycarbonyl, alkoxycarbonyloxy, hydroxycarbonyl, halide,cyano, amide, carbamoyl, thiocarbamoyl, ureido and thioureido.Heteroalkyl refers to alkyl groups (or rings) that contain at least oneheteroatom selected from N, O, and S, wherein the nitrogen and sulfuratoms are optionally oxidized, and the nitrogen atom(s) are optionallyquaternized. A heteroatom can form a double bond with a carbon atom. Aheteroalkyl group can be attached to the remainder of the moleculethrough a/the heteroatom. Heteroaryl refers to aryl groups that containfrom one to five heteroatoms selected from N, O, and S, wherein thenitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. In preferred embodiments of theabove, R¹, R², R⁷, and R⁸ are each hydrogen or C₁-C₃ alkyl. In someembodiments, only one of the R³ to R⁶ members is a functional group.

A functionalized parylene membrane may be covalently modified via thefunctional group thereof to facilitate the detection or retention of thecell or particle to be captured. In some embodiments, the functionalizedparylene membrane filter is further modified by having an antibody orantibody fragment covalently or non-covalently bound thereto. In someembodiments, the antibody or fragment may specifically recognize thecell to be isolatd. In other embodiments, the surface of thefunctionalized parylene membrane filter is covalently or non-covalentlyattached to a ligand capable of binding a receptor on the surface of acell to be retained by the filter. The conjugation chemistry describedabove can be used in attaching ligands or antibodies to thefunctionalized parylene membrane filter.

Preferred types of parylene include commercially available parylene C,F, A, AM, N, and D. Of the three most common types of parylene shownbelow, parylene C is perhaps the most widely used in industry. Furtheradvantages of the parylene membrane substrate include strength andflexibility (e.g., Young's modulus≈4 GPa), conformal pinhole-freeroom-temperature deposition, low dielectric constant (≈3) high volumeresistivity (>10¹⁶ Ω-cm), transparency, and ease of manipulation usingstandard microfabrication techniques such as reactive ion etching (RIE).

In other embodiments, the invention provides parylene membranefiltration devices which comprise the parylene membrane filter and ahousing. Advantageously, the parylene membrane filter allows thefiltration of many fluids, including, biological fluids as described indetail below.

As used herein, the term “monodispersed” refers to openings or holes onthe membrane filter having substantially identical size, dimension andshape.

FIG. 1 illustrates a fabricated parylene membrane filter according tothe invention 100. As shown, the membrane filters having predetermineddifferent geometric designs can be fabricated to achieve the desiredresults. A geometric design includes the plan of size, shape and densityof the openings on the membranes. FIG. 1A shows a parylene membranehaving 50 parylene membrane filters formed therein. A representativemembrane filter 110 is shown. As set forth above a variety In oneembodiment, square holes are monodispersed. In another embodiment, theholes are uniformly-spaced. FIG. 1B shows a portion of one of themembrane device having circular openings 125. In one embodiment, thecircular holes are monodispersed. In another embodiment, the holes areuniformly-spaced. FIG. 1C shows a portion of one of the membrane devicehaving oval holes 135. In one embodiment, the oval holes aremonodispersed. In another embodiment, the holes are uniformly-spaced.The shape of the holes on the membrane includes, but is not limited to,a circle, an oval, a symmetric polygon, an unsymmetrical polygon, anirregular shape and combinations thereof. In some embodiments, themembrane filter has circular, rectangular, or hexagonal holes.

FIG. 1B illustrates a membrane having monodispersed and uniformly spaced10 micron circular holes. FIG. 1C illustrates a membrane havingmonodispersed and uniformly-spaced 8 micron×14 micron oval holes. Thehole size can be precisely controlled using reactive ion etching (RIE)technology (see, U.S. Pat. No. 6,598,750 incorporated herein byreference). Hole dimensions and opening factors can vary from 0.1 micronto 12 micron or greater and from 4 to 45%, respectively. Preferably, insome embodiments, the hole dimensions are from 5 micron to 12 micron. Insome embodiments, the density of the holes can be precisely controlledup to 40,000 holes per square millimeter. The thickness of the parylenemembrane can also be controlled. Generally, a thicker membrane is neededfor a higher density of holes. Parylene membranes with variousthicknesses from about 0.1 micron or 1 micron to 15 micron or more arecontemplated. In one embodiment, a 10 micron thick parylene membrane isused.

In some embodiments, parylene membrane filters having uniformly-spacedholes are prepared. In other embodiments, parylene membrane filtershaving an array of monodispersed holes are prepared. The membranes withmonodispersed and uniformly distributed holes have a higher cellcapturing efficiency than membranes with randomly distributed andpolydispersed holes.

FIG. 2 illustrates one aspect of a parylene membrane filter device 200.The device includes a parylene membrane 250 mounted inside a housing.The housing can adopt a variety of sizes and shapes, which include, butis not limited to, tubular, spherical and cubical shapes. In oneembodiment, the housing is made of a top chamber 220 having an insertionport 270 and a bottom chamber 230 having an exit port 280. Variousmaterials can be used for the construction of the chambers. Thematerials include, but are not limited to, polysiloxane, polycarbonate,polyacrylate, polyethylene, polystyrene, polysaccharides and copolymersand combinations thereof. In one embodiment, the material used forconstruction of the chambers is polydimethylsiloxane (PDMS). In oneembodiment, the top chamber, the bottom chamber and a parylene membraneare clamped by two pairs of jigs (210 a, 210 b; 240 a, 240 b). The topjig 210 a and 210 b can be made of polyacrylate, polyketone,polystyrene, polypropylene and the like. The bottom jig is made of anengineering material, which includes, but is not limited to, apolyketone, a polysulfone, a polysulfide or a polyimide. In oneembodiment, the bottom jig is polyetheretherketone (PEEK). The jigs areheld together by suitable means 260 and 265, such as bolts, fasteners,screws, latches, links, joints, locks or unions.

In some embodiments, the membrane filter is any membrane filter setforth in the detailed description of the specification of U.S. patentapplication Serial No.: (yet to be assigned) filed this same date andentitled “MEMBRANE FILTER FOR CAPTURING CIRCULATING TUMOR CELLS” andhaving attorney docket no. 020589-008610US, which is particularlyincorporated herein by reference in its entirety, with respect to suchmembrane filters and also the suitable membrane substrates thereof.

Various parylene and parylene-like materials can be used as substratesin the present invention when circulating tumor cells are to bedetected. In this application, other materials, such as polyimide,polysiloxane, polyester, polyacrylate, cellulose, Teflon andpolycarbonate may also be suitable filter substrates. In thisapplication, the substrates used in the present invention are notlimited to materials discussed above, but also include other materials,which perform substantially the same function as parylene, insubstantially the same way as parylene and achieve substantially thesame result as parylene. Preferably, these have a figure of merit ofabout 890 and/or having a Young's modulus≈4 GPa. Figure of meritprovides a measure of the efficiency of the filtration device. A largefigure of merit number is an indication of higher filtration efficiency.Figure of merit is defined as the recovery rate divided by time.Recovery rate is defined as particles recovered divided by the totalnumber of target particles. The time used in the calculation of figureof merit is the total processing time to conduct the testing.

In another embodiment, filters having multiple membranes with variousgeometric designs can be used for the separation of CTC cells and othercells and particles in the blood. Each successive membrane can have thesame or different geometric designs. For example, the size, shape anddensity of the holes can be varied at each membrane layer. An activesandwich layer can be optionally placed in between any two membranefilters. Alternatively, the sandwich layer can be an inert layer. FIG. 3illustrates an embodiment of a two-membrane filter device 300. The topfilter 350 and the bottom filter 355 are parylene or parylene-likemembranes. The top filter and the bottom filters can have the same ordifferent geometric designs. In one embodiment, the top filter hascircular holes and the bottom filter has oval holes. In anotherembodiment, the dimension of the holes on the top filter isapproximately 10 micron and the dimension of the holes on the bottomfilter is 8 by 14 micron. In a preferred embodiment, each membrane hasan array of monodispersed holes. In another preferred embodiment, eachmembrane has an array of uniformly-spaced holes. The parylene membranes355 and 355 are mounted inside a housing. The housing can adopt avariety of sizes and shapes, which include, but is not limited to,tubular, spherical and cubical shapes. In FIG. 3, the housing is made ofa top chamber 320 having an insertion port 370 and a bottom chamber 330having an exit port 380. Various materials can be used for theconstruction of the chambers. The materials include, but are not limitedto, polysiloxane, polycarbonate, polyacrylate, polyethylene,polystyrene, polysaccharides and copolymers and combinations thereof. Inone embodiment, the material used for construction of the chambers ispolydimethylsiloxane (PDMS). In one embodiment, the top chamber, thebottom chamber and a parylene membrane are clamped by two pairs of jigs(310 a, 310 b; 340 a, 340 b). The top jig 310 a and 310 b can be made ofpolyacrylate, polyketone, polystyrene, polypropylene and the like. Thebottom jig is made of an engineering material, which includes, but isnot limited to, a polyketone, a polysulfone, a polysulfide or apolyimide. In one embodiment, the bottom jig is polyetheretherketone(PEEK). The jigs are held together by suitable means 360 and 365, suchas bolts, fasteners, screws, latches, links, joints, locks or unions.

In yet another embodiment, the present invention provides a membranefilter device having a plurality of membrane filters assembled inside ahousing for the separation of fine particles, such as, bacteria orviruses. Each of the upper membranes has a different geometric designfrom each of the adjacent lower membranes. The geometric design variesin the hole size, shape and density. The holes on each of the membranesare designed such that each successive layer only traps the desiredcells or particles and allow undesired substance to pass through. Forexample, cells or bacteria passing through a first filter can becaptured by the subsequent second or the third filter having a differentgeometric design.

In another aspect, the membrane substrate for use according to theinvention can also be a polyimide. In another embodiment, at least oneof the plurality of membrane filters comprises a parylene membranefilter.

FIG. 4 illustrates the SEM images of the filter device of the presentinvention (FIGS. 4B-4D). FIG. 4A reveals randomly distributed holes andfused holes in the commercial filter device. The device of the presentinvention have a predetermined geometric design with an array ofprecisely controlled holes 425, 438 and 455, which have allowed anefficient and successful trapping of the tumor cells 435 and 458 (FIGS.4B-4D). In one embodiment, prostate cancer cells are captured by themembrane filters.

The device of the present invention can be fabricated using spinningcoating technology known in the art. A photoresist material is firstspin-coated on a silicon wafer. Next, a thin layer of substrate isdeposited on top of the photoresist material. Finally, patterning isgenerated by reactive ion etching (RIE). Alternatively, a thin layer ofsubstrate can be directly deposited on top of a silicon wafer. The filmis released using water or an organic solvent. Common organic solventssuitable for releasing the film include, but are not limited to,ethanol, acetone, tetrahydrofuran, dichloromethane, chloroform, C₁-C₈hydrocarbon solvents. Parylene membranes can be prepared according to adeposition technology (see, U.S. Pat. No. 6,598,750). In someembodiments, the photomaterial used is AZ1518; the substrate is aparylene, the preferred substrate is parylene-C. Parylene membranefilters as large as 8×8 square millimeters can be fabricated.

The devices for use according to the invention also provide a pressurethat can be optionally applied to the fluid to facilitate the filtrationprocess. In one embodiment, the pressure applied to the fluid isgenerated by gravity. In another embodiment, the pressure applied to thefluid is generated by an electrokinetic, for example, electroosmosis,and a ratchet pump. In yet another embodiment, fluid pressure isgenerated using pneumatic or magneto hydrodynamic pumps. In yet afurther embodiment, the pressure applied to the fluid is generated by amechanical device. One example of a useful mechanical pressuregenerating device is a screw-type pumping device or a peristaltic pump.

CTCs are diagnostically as well as prognostically critical as they areassociated with clinical stage, disease recurrence, tumor metastasis andpatient survival following therapy. Special considerations are neededfor designing a microfabricated device to address the extremely lownumber of CTCs in blood (on the order of 1 per >1010 blood cells), andlarge sample volume required (5-7.5 ml of whole blood). Since theaverage size of CTCs for most common epithelial cancers is significantlylarger than most blood cells, separation based on size can be veryeffective (Vona, G. et al., American Journal Of Pathology 156: 57-63(2000)). While the membrane filter allows processing large samplevolumes of blood; parylene provides a unique filter substrate due to itsflexibility and biocompatibility.

Accordingly, in some preferred embodiments, the filters and devicesaccording to the invention are be used in the capture, detection andcharacterization of subclinical tumor cell deposits in peripheral blood.These particular inventive methods can be used by oncologists to developestimates of the risk of recurrence of a cancer for an individualpatients, to tailor therapies more effectively and to monitor responseto therapy. One of the most important determinants of prognosis andmanagement of cancer is the absence or presence of metastaticdissemination of tumor cells at the time of initial presentation andduring treatment (see, Lugo T G, et al., J Clin Oncol. 21(13):2609-15(2003)). This early spread of tumor cells to lymph nodes or bone marrow(BM) is referred to as “disseminated tumor cells” (DTC), or ascirculating tumor cells” (CTC) when in the peripheral blood (PB). It hasbeen well established that these DTC or CTC can be present even inpatients who have undergone complete removal of the primary tumor, andthat this phenomenon is the basis for the later development of overtmetastases in these patients. Indeed, the possible presence of earlytumor dissemination is the rationale behind the use of systemic adjuvantchemotherapy in patients who have undergone definitive treatment of theprimary tumor (Schabel F M, Jr., Cancer 39(6 Suppl):2875-82 (1977)). Thedetection of DTC has proven to be a useful tool in determining thelikelihood of disease progression (Braun S, N Engl J Med. 353(8):793-802(2005)).

The current techniques used for DTC and CTC capture and identificationhave significant barriers including multiple procedural steps, handlingof relatively large volumes of sample, substantial human intervention,extremely high cost and importantly, the lack of reliability andstandardization for the detection methods, which have to date been basedon positive markers of tumor cell detection. Whereas the detection ofDTC by BM aspiration involves an invasive procedure (and hence far morelikely to cause complications and lack of compliance from both patientsand physicians), PB sampling offers a relatively less invasive option.Therefore, although the rates of DTC detection in the BM far exceedthose for CTC detection in the PB, the investigation of PB as a targetcompartment is receiving more attention in recent years (see, Pierga JY, et al., Clin Cancer Res. 10(4):1392-400 (2004); and Redding W H, etal., Lancet 2(8362):1271-4 (1983)).

One of the reasons of lesser rate of detection of CTC in blood could bethe limitation of the current methodologies, which are sub-optimal tocapture CTC from blood. Development of methods such as flow-cytometry,magnetic cell separation and di-electrophoresis has been proposed toincrease the yield in the PB (see, Gilbey A M, et al., J Clin Pathol.57(9):903-11 (2004)). Although in development for many years, thesemethods are still available essentially only in the research setting,their clinical applicability largely restricted due to expensiveautomation and lack of efficiency in PB. Therefore, it is clear that thedevelopment of a device capable of detecting the earliest metastaticspread of tumor in the PB (CTC) can revolutionize the approach to thedisease management. Indeed, in addition to being a predictor of diseaseprogression, detection of circulating tumor cells (CTC) in PB has beenshown to be an important factor in therapeutic monitoring in patientsreceiving therapy for metastatic breast cancer (Cristofanilli M, et al.,N Engl J. Med. 351(8):781-91 (2004) and Cristofanilli M, et al., J ClinOncol. 23(7):1420-30 (2005)).

As discussed, BM as a source for detection of DTC has been documentedfor higher frequency of positive samples as compared to PB (see, PiergaJ Y, et al., Clin Cancer Res. 10(4):1392-400 (2004) and Redding W H, etal., Lancet. 2(8362):1271-4 (1983)). The presence of DTC in the BM hasbeen proven to be clinically significant in a variety of tumors (seeTable 2 for summary of published data for breast cancer, and table 3 forlung cancer). The collection of BM however, is problematic and requiresspecial invasive procedures that may be unpleasant for the patient andinconvenient for the physician.

TABLE 2 Breast cancer and bone marrow tumor cells DT: disseminated tumorcells DTCBM−: no DTC to bone marrow; DTCBM+: DTC to bone marrow*estimated 2 years recurrence rate

TABLE 1 Breast cancer and bone marrow tumor cells Clinical Follow- # up% Patients Recurring (number) Patients (years) DTCBM− DTCBM+ p-valueDearnaley, 39   9.5 31% (8/26) 85% (11/13) <0.05 Mansi, 350   2.3 25%(64/261) 48% (43/89) <0.05 1991 Cote, 49  2* 16% (5/31) 54% (7/13) <0.041991 Diel, 211 2  3% (4/130) 27% (22/81) 0.0001 1992 Diel, 727 3  8%(34/412) 35% (109/315) <0.001 1996 Braun, 552 4  8% (28/353) 39%(79/199) 2000 Gebauer, 393 6 20% (46/227) 35% (59/166) <0.001 2001Gerber, 554   4.5  9% 24% 0.0001 2001 DT: disseminated tumor cellsDTCBM−: no DTC to bone marrow; DTCBM+: DTC to bone marrow *estimated 2years recurrence rate Dearnaley DP, et al., Eur J Cancer 27(3): 236-9(1991). Mansi JL, et al., Eur J Cancer 27(12): 1552-5 (1991). Cote RJ,et al., J Clin Oncol. 9(10): 1749-56 (1991). Diel IJ, et al., J ClinOncol. 10(10): 1534-9 (1992). Diel IJ, et al., J Natl Cancer Inst.88(22): 1652-8 (1996). Braun S, et al., N Engl J Med. 342(8): 525-33(2000). Gebauer G, et al., J Clin Oncol. 19(16): 3669-74 (2001). GerberB, et al., J Clin Oncol. 19(4): 960-71 (2001).

TABLE 3 Lung cancer and bone marrow tumor cells No Patients inAntibodies study No Patients DTCBM+ (%) Non-small cell lung cancer:Pantel, 1993 CK18 82 18 (22%) Cote, 1995 CK 43 17 (40%) Pantel, 1996CK18 139   83 (59.7%) Ohgami, 1997 CK18 39 15 (39%) Small cell lungcancer Leonard EMA, CK 12  8 (67%) Pantel K, et al., Lancet 347(9002):649-53 (1996). Ohgami A, et al., Ann Thorac Surg. 64(2): 363-7 (1997).Leonard RC, et al., Cancer Res. 50(20): 6545-8 (1990).

Peripheral blood (PB) acts as a vector carrying the tumor cells to everyanatomic site. Since tumor cell dissemination is considered an earlyevent in the multi-phasic metastatic process, the prospect of detectingtumor cells in the PB before clinical evidence of distant, overtmetastasis has been of interest for over a decade. Unfortunately, theyield of CTC from PB is extremely low (see, Pierga J Y, et al., ClinCancer Res. 10(4):1392-400 (2004) and Redding W H, et al., Lancet.2(8362):1271-4 (1983)). Redding et al. found that 28.2% patients withbreast cancer showed extrinsic cancer cells in their BM, but only 2.7%of these patients had detectable cells in their PB. Most of the studieslooking for the presence of early metastases in the blood have focusedon using molecular methods. Nonetheless, many studies have sought to useICC as well for detection of CTC in PB (see, Bischoff J, et al., RecentResults Cancer Res. 162:135-40 (2003), and the need of using enrichmenttechniques has become more apparent.

However, enrichment techniques are time consuming and there is noguarantee that cells will not be lost in the processing. Therefore, aprocedure that directly extracts cells from PB with minimal proceduralsteps is vital to make this prognostic indicator practical in theclinical setting. Accordingly, in one aspect this invention provides aparylene membrane filter, device, method, and/or an integrated systemwhere a parylene filter membrane is located in a device in which cellfractionation bypasses the need for gradient separation. This allowsincreased cellular yield from PB.

We analyzed PB samples from 133 patients using other methods with stageII, II or IV breast cancers. This preliminary data show, that thefrequency of DTC and CTC is generally low. Even in metastatic breastcancer in BM aspirates the incidence was 40%. Our data further supportthe findings by Redding et al. (Redding W H, et al., Lancet.2(8362):1271-4 (1983) that the frequency of DTC is higher in BM than inPB. These findings indicate the need for effective enrichment methods.

Number of Patients Positive/ Number of Patients Stage BM PB II 0/30 (0%)0/30 (0%) III 4/65 (6%) 1/65 (2%) IV 15/38 (40%)  5/38 (13%) Incidenceof tumor cell samples from patients with breast cancer who had both bonemarrow and peripheral blood tested by stage.

In preferred embodiments, the parylene membrane filters are used todetect CTCs in PB from patients with prostate, bladder and breastcancer. The detection can be used in the diagnosis and prognosis ofthese conditions or in evaluating the response to therapy. Breast Canceris the most commonly diagnosed form of cancer and is the second mostcommon cause of cancer related death in women. Although increasedpatient awareness and improved screening techniques now permit earlydetection of localized and resectable tumors, many women still die fromrecurrent breast carcinoma suggesting that a substantial number ofpatients already have distant DTC at the time of diagnosis. The clinicalsignificance of DTC to the BM has long been established by us as well asothers (see, Braun S, N Engl J. Med. 353(8):793-802 (2005) and Cote R J,et al., J Clin Oncol. 9(10):1749-56 (1991) (3, 20)

Prostate cancer is the most commonly diagnosed form of cancer in man,and is the second most common cause of cancer related death in men,second only to lung. Overall, 99% of men diagnosed with prostate cancersurvive at least 5 years. For the men whose cancer has already spread todistant parts of the body when it is found, 34% will survive at least 5years. Despite substantial treatment options, there is still a need forimprovement. The clinical relevance of DTC and CTC has been shown inmany prior studies (see, Freeman J A, et al., J Urol. 154(2 Pt 1):474-8(1995); Weckermann D, et al., J Clin Oncol. 17(11):3438-43 (1999);Allard W J, et al., Clin Cancer Res. 15; 10(20):6897-904 (2004); andChen et al., Urology 65(3):616-21 (2005)).

Bladder cancer: Transitional cell carcinoma is the second most commongenitourinary malignancy in US and third most common cause of deathamong genitourinary tumors. Only a proportion of patients at risk willrespond to therapy. The significance of occult metastatic spread ofbladder cancer in clinical management of the disease has been recognizedfor over a decade now. The 5-year survival rate for bladder cancer(which reflects overall cure rates) is proportional to the pathologicalstage but is approximately 50%, with patients dying from metastaticdisease. (see, Lerner S P, et al., Urol Clin North Am. 19(4):713-23(1992) and Hofmann T, et al., J. Urol. 169(4):1303-7 (2003)).

Many markers can be used in a multimarker analysis of breast cancerspecimens. Breast cancer represents a much studied disease forexpression of different markers on CTC. For instance, the presence ofhormone receptors for estrogen (ER) and progesterone (PR) in primarytumors has been shown to be positively correlated with outcome of breastcancer patients and are currently among the strongest prognostic factorsof breast cancer. Her 2 neu: HER-2/neu is a proto-oncogene that encodesa transmembrane receptor belonging to the family of epidermal growthfactor receptors. Her-2/neu (Her-2) overexpression, usually attributableto HER-2 gene amplification, occurs in 20-25% of breast cancer patientsand is associated with a poor prognosis. Recently, it has beendemonstrated, that even >than 30% of patients with no Her-2 neuoverexpression in primary tumor acquire this characteristic on CTC andindicate the need of targeted therapy. The putative breast cancer stemcell phenotype: has been defined as CD 44+CD24−/low. Cells with thisphenotype have been identified as cells capable of self-renewal andtumorigenicity. The hypothesis that solid tumors raise from tumor stemcells has gained on importance in recent years, and superficial markers,which are reflecting the phenotype of putative stem cells, can be usedto enrich the putative stem cell population, to further characterizethese cells.

Stem cell antigen-1 (Sca 1): Stem cell antigen 1 (Sca-1) appears to bepreferentially expressed in mammary stem and/or progenitor cells. Thereis evidence for upregulation of Sca 1 in breast cancer. Sca 1 can alsobe used to enrich for murine prostate cells capable of regeneratingtubular structures containing basal and luminal cell lineages in adissociated cell prostate regeneration system.

Proliferation and apoptosis markers can also be used according to themethods of the invention. Survivin: Survivin expression has beenupregulated in cancer tissues, and present in different cancer celllines. The role of survivin has been connected with prolonged survivalof cancer cells and resistance to therapy. It is member of inhibitorapoptosis family. It may even play an important role in carcinogenesis.M30: M30 is an antibody that has been shown to detect fragmented CK 18,a characteristic of apoptotic cells, and has been used to demonstrateapoptosis in tumor tissue. Ki67: Ki67 is an antigen that representsproliferating cells. Circulating tumor cells have been shown to have lowlevel of Ki67 expression, similarly to DTC in BM. Given the possibilityof sub-populations within CTC, this marker may be useful incharacterizing and profiling CTC, especially as a member of a panel ofmarkers.

A variety of immunocytochemistry (ICC) techniques may be used to DTC andCTCs. For instance, immunological detection of occult metastatic cellscommonly employs antibodies specific for low-molecular weightcytokeratin (CK) proteins to distinguish the epithelial tumor depositsfrom normal lymph node elements and BM or PB. A cocktail of two anti-CKantibodies, AE1 and CAM5.2 (which in combination recognize thepredominant intermediate filament proteins in simple epithelial cells)can be used (see, Chaiwun B, et al., Diag Oncol 2:267-76 (1992)). One ofthe primary difficulties with current ICC techniques for detecting DTCis that they are labor intensive and time-consuming, factors that maylimit their general availability. In addition, the technology is notamenable to assess multimarkers on a single cell.

In addition, Quantum Dot (QD)-based ICC may be used. The most recentwork with QDs and spectral imaging has broadened the utility of ICC byenable multimarkers to be assessed on a single cell (see, Jaiswal J K,et al., Nat Methods. 1(1):73-8 (2004)). QDs can be made from a varietyof inorganic compounds (Gao X, et al., Curr Opin Biotechnol. 16(1):63-72(2005)). The QD nanocrystals usually used for ICC are made from a 10-nmcadmium and selenium (CdSe) core that is then coated with asemiconductor layer (ZnS) to improve the optical qualities of thematerial. This core and semiconductor layer particle is then coated withan additional polymer shell. This additional outer polymer shell enablesthe nanocrystal to be conjugated with a biological molecule at the sametime maintaining their optical properties. These QD conjugates aremultivalent such that more than one biomolecule can be attached to asingle QD. Compared to immunofluorescent dyes, QDs are brighter, notprone to photobleaching, come in a wide range of colors, and theiremission can be tuned to any desired wavelength by modulating the sizeof the particle. The QDs also have narrow emission spectra enabling morecolors to be used with minimal channel overlap, and multiple colors canbe simultaneously emitted by a single light source. The properties ofQDs have been reviewed by Watson A, et al., Biotechniques 34(2):296-300,2-3 (2003).

QDs have been heralded as among the most promising techniques forcellular imaging, however, there were questions whether QDs couldspecifically and effectively label molecular markers at the subcellularlevel. QD-based probes are effective in cellular imaging in multiplextarget detection. Spectral imaging technology can further enhance theutility of QDs in ICC. This technology, described later in greaterdetails is based on color differentials such that a different chromogenis used for each of the many markers tested, and a specially designedsoftware serially “erases” a given chromogen from the microscope imageto assess each marker on an individual basis, even when the colordifferences are not discernable to the naked eye. This technology canprovide information on multiple marker status on specific individualcells.

A potentially more sensitive molecular approach for detection of DTC isthe reverse-transcriptase polymerase chain reaction (RT-PCR), which hasbeen applied to several malignancies employing a variety of markertranscripts as targets. Since the first study by Smith and colleagues in1991, many authors have reported molecular diagnoses in the lymph nodes,blood, and BM in cancer patients. Application of RT-PCR in regional andsentinel lymph nodes has been described for a number of cancers,including melanoma, colorectal cancer, and cancers of prostate, breastand lung (see, Smith B, et al., Lancet 338(8777):1227-9 (1991); ShariatS F, et al., J Urol. 170(3):985-9 (2003); Sakaguchi M, et al., Ann SurgOncol. 10(2):117-25 (2003); Wallace M B, et al., Am J Respir Crit CareMed. 167(12):1670-5 (2003); Blaheta H J, et al., J Invest Dermatol.114(4):637-42 (2000); Shariat S F, et al. J Clin Oncol. 22(6):1014-24(2004)) and Corradini P, et al., Ann Oncol. 12(12):1693-8 (2000)). Manyof these compare the ICC-based detection with RT-PCR for sensitivity andconclude that RT-PCR may provide enhancement in detection, provided thetarget markers are sufficiently specific. Various formats of RT-PCRassays (see, Burchill S A, et al., Br J Cancer 71(2):278-81 (1995);Aquino A, et al., J Chemother. 14(4):412-6 (2002); and Wiedswang G, etal. J Clin Oncol. 21(18):3469-78 (2003)) have also been used fordetection of DTC in BM in patients with cancers of breast, colon, lung,etc. With some exceptions of organ-specific markers like maspin ormammaglobin for breast cancer or uroplakins for bladder cancer most ofthe molecular targets used in these RT PCR assays have been shown tolack the requisite specificity due to illegitimate expression innon-target hematopoietic cells as shown by us and many otherinvestigators (see, Grunewald K, Lab Invest. 82(9):1147-53 (2002);Bostick P J, et al., N Engl J Med. 339(22):1643-4 (1998); Osman I, etal. Int J Cancer 111(6):934-9 (2004); Pelkey T J, et al., Clin Chem.42(9):1369-81 (1996); Raj G V, et al., Cancer 82(8):1419-42 (1998); andGhossein R A, et al., Clin Cancer Res. 5(8):1950-60 (1999)). RT-PCR hasalso been used to enhance the sensitivity of detection in PB in avariety of cancers including prostate cancer, breast cancer,gastrointestinal tract cancers, melanoma, colorectal cancers,head-and-neck cancer, etc. (see, Partridge M, et al., Clin Cancer Res.;9(14):5287-94 (2003); Palmieri G, et al., J Clin Oncol. 21(5):767-73(2003); Yokoyama S, Yamaue H. Arch Surg. 137(9):1069-73 (2002); and Li SM, et al., J. Urol. 162(3 Pt 1):931-5 (1999)) although the same concernsof non-specificity due to the illegitimate transcription of target genesin the non-target hematopoietic cells as described above exist, whichhave hampered the use of these assays in routine clinical diagnostics.

The identification of tumor cells by ICC has traditionally beencomprised of a single marker or a cocktail of markers that identifyepithelial cells. All markers used to co-express to some degree onhematopoietic cells present in the samples. This has led to subjectivityand disagreement in assessing for the presence of tumor cells. Positiveand negative selectors address this issue. The positive markers identifythe epithelial cells, while the negative markers identify thehematopoietic cells. The tumor cells can be determined to not co-expressthe hematopoietic markers. In this way all cells that express thenegative selector can be classified as non-tumor cells, regardless ofwhether or not they express the positive selector (FIG. 13 in resdesign). ICC using negative and positive selectors identifies falsepositives and greatly increases the selectivity over traditional singlepositive selection ICC. Previously, false positives were distinguishedfrom true positives on the basis of morphology alone, a subjectiveassessment that is difficult in these types of cell preparations.

A variety of epithelial antigens (Positive Selector), as known to one ofordinary skill in the art, are suitable. For instance, Cytokeratins (CK)are intermediate filament proteins which are normal components ofepithelial cytoskeleton, and hence are commonly used to identifycarcinoma in non-epithelial compartments. The antibody cocktailtypically used in ICC detection of DTC is raised against peptides fromboth acidic and basic CK; such as the combination of antibodies AE-1(reactive with CK-10, 14, 16 and 19) and CAM 5.2 (reactive with CK-7 andCK-8) used by us ((64) see Appendix) and many other investigators (27,64-66).

Epidermal Growth Factor Receptor (EGF-R) can also be used.Over-expression of epidermal growth factor receptor (EGF-R, product ofc-erbB-1 proto-oncogene) is associated with poor prognosis in patientswith cancer, and identifies breast, bladder, gastric, cervix, ovary andNSC lung cancers, making it a significant prognosticator. Since EGF-R isnot expressed in hematopoietic cells, it is a particularly suitablemarker for detection of circulating tumor cells in cancer patients.

Epithelial Cell Adhesion Molecule (EpCAM) is also suitable. EpCAM isknown variously as Human Epithelial Antigen (HEA), BerEP4: GA-733.2 or17-1A, antibodies to this surface antigen show a very broad pattern ofreactivity with human epithelial tissues from simple epithelia to basallayers of stratified non-keratinized squamous epithelium and epidermis.It does not react with mesenchymal tissue, including lymphoid tissue.

A variety of hematopoietic antigens (Negative Selector) are alsosuitable: CD 45 is a hematopoietic cell marker for ICC: CD 45 is alsoknown as leukocyte common antigen (LCA) precursor and is a receptorglycoprotein that ranges in size from 180 to 220 kDa. The antigen ispresent on the surface of all human leukocytes. An antibody to CD45immunoreacts with the majority of white blood cells (WBCs) present inthe blood and BM (75). CD 68 is a primarily intracellular 110 kDAlysosomal glycoprotein, which can also be found on the surface ofmacrophages, monocytes, neutrophils, eosinophils and large lymphocytes.

In addition Tumor-/Tissue-restricted markers are also suitable.Mammaglobin: The mammaglobin gene is a human breast cancer-associatedgene. The gene encodes a 10 kDa glycoprotein and is distantly related toa family of epithelial secretory proteins. Mammaglobin is amammary-specific member in the uteroglobin family and is known to beoverexpressed in human breast cancer. It, along with Maspin, iscurrently under investigation at the transcript level as a potentialmarker to detect circulating tumor cells in the PB from patients withbreast cancer.

PSA: Prostate Specific Antigen (PSA) is the classical indicator fortransformed prostate tissue. It is a serine protease that hydrolyzes themajor seminal protein, the seminal plasma mobility inhibitor precursor,or semenogelin I, which leads to semen liquification. Serum PSA levelabove 4 ng/ml is an aid in the early detection of prostate cancer. PSAexpression is useful in detection of DTC/CTC.

Uroplakin Family Markers—Molecular markers specific for a single type ofepithelium are rare. Bladder epithelium is unique in its expression offamily of transmembrane proteins uroplakins (UP). UP expression can betracked at both RNA and protein levels in primary bladder cancer tissuesas well as metastatic legions. The expression of UP transcripts incirculating and metastatic urothelial cancer cells can be detected.Uroplakin II can be a specific marker for the assessment of perivesicalextension and lymph node status after radical cystectomy and, at thetranscript level, for detection of urothelial cancer cells in PB. UPIItranscripts are a preferred marker for bladder cancer specimens.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Parylene Membrane Performance

The design, fabrication and testing of a parylene membrane filter deviceaccording to the invention is described. Even without much optimization,the device demonstrated 89% recovery and 9 log 10 enrichment toout-perform most current methods used in the field (see, Lara, O., etal., Experimental Hematology, vol. 32: 891-904 (2004)). Moreover, lessthan 10 minutes can be required for each sample separation, compared tocurrent multistep processing needing more than 1 hour.

Two filters as shown in FIG. 1 were tested in this initial study. Thefirst parylene membrane filter had an array of circular holes with a 10micron diameter and the second parylene membrane filter had an array of8 by 14 micron oval holes. To fabricate these membrane filters,photoresist AZ1518 was spin-coated on silicon wafer, followed by 10micron parylene-C deposition and patterning with RIE. Finally the wholefilm was acetone released overnight. The individual membrane filter wassandwiched between two PDMS chambers and clamped tightly with a jig(FIG. 2). FIG. 3 compares the SEM pictures of commercially availablepolycarbonate membrane filter used for rare tumor cell detection (FIG.3A) and these two microfabricated parylene membrane filter (FIG. 3B,C,D). The commercial membrane filter shows randomly and sparselydistributed holes, with many noticeably fused, resulting in largeropenings and reduced fidelity.

For testing, a first syringe was each inserted into the top portaccessing a first PDMS chamber and a second syringe was inserted intothe bottom port accessing a second PDMS chamber. Sample was loaded usingthe top syringe and dispensed manually to traverse the filter. Theflow-through is collected by the bottom syringe. Further rinses withbuffered saline were performed similarly.

The filtration of prostate cancer cells was conducted by inserting eachsyringe into ports of the top and the bottom chambers as described. Asample was loaded using the top syringe and dispensed manually totraverse the filter. The filtrate was collected by the bottom syringe.The prostate cancer cells were cultured cells derived from humanmetastatic prostatic adenocarcinoma (LNCaP). These cells were stainedwith hematoxylin, and serially diluted in buffered saline to the desirednumbers for device testing. The average diameter of LNCaP cells wasmeasured to be 19 microns×3 microns.

Parylene membrane filters having both circular and oval holes weretested. The recoveries for circular and oval designs were 87.3%±7.0% and89.1%±7.0%, respectively (Tables 1 and 2). For detection limit tests,the cell numbers were lowered to less than 10 cells per milliliter.Tables 3 and 4 demonstrate that the device can capture as few as 4 tumorcells per milliliter.

TABLE 1 Recovery Test for Circular Hole Design 1 2 3 4 5 Tumor CellsAdded 402 402 402 402 402 Cells Recovered 346 400 344 327 339 Cells inFlow-Through 2 1 0 1 0

TABLE 2 Recovery Test for Oval Hole Design 1 2 3 4 5 Tumor Cells Added86 86 86 86 86 Cells Recovered 79 84 74 78 68 Cells in Flow-Through 0 00 0 0

TABLE 3 Capture Limit Test for Circular Hole Design 1 2 3 4 5 TumorCells Added 4 4 4 4 4 Cells Recovered 4 3 3 3 3 Cells in Flow-Through 00 0 0 0

TABLE 4 Capture Limit Test for Oval Hole Design 1 2 3 4 5 Tumor CellsAdded 8-9 8-9 8-9 8-9 8-9 Cells Recovered 7 7 6 9 7 Cells inFlow-Through 0 0 0 0 0

The device performance was also tested by spiking known numbers of LNCaPcells in peripheral blood from healthy donors. The captured cells werecaptured on the parylene membrane filter (see, FIG. 5). Similar toexperiments with buffered saline, the oval filter design resulted in89.0%±9.5% recovery from blood (Table 5):

TABLE 5 Recovery for Tumor Cells Spiked in 1 ml Whole Blood with OvalHole Design 1 2 3 4 5 Tumor Cells Added 41 41 41 41 41 Cells Recovered33 35 42 39 34 Cells in Flow-Through 0 0 0 0 0

Example 2 Capture and Detection of Bladder Cancer Cells in Urine

Background to the Example: Bladder cancer is an important public healthproblem. With its incidence continuing to increase, bladder cancer isnow the fifth most common cancer in US, with an estimated 56,500 newcases predicted for 2002, of which 12,600 patients are expected to dieof the disease. Cystoscopy is an invasive and relatively costlytechnique for detecting bladder cancer. The method can be inconclusiveat times, particularly in cases of cystitis. A simpler, noninvasiveassay for detecting recurrence of bladder cancer is needed.

A clinically useful urinary marker assay should be easy to perform, haveminimum requirements for sample processing and be highly sensitive andspecific in diagnosis. Urinary cytology can serve as an excellent testfor screening because it is simple, non-invasive and inexpensive; but,in general, experience in the field has generally demonstrated thelimitations of urinary cytology in detecting tumors, especially in lowgrade tumors in which the sensitivity varies. When others have evaluatedthe performances of the combination of ultrasound and urine cytologyversus cystoscopy in the follow-up of bladder tumors, they found thecombination to be sufficient for systematic surveillance in superficialtumors or low-grade bladder tumors as an alternative to cystoscopy.

Urine cytology is traditionally used to detect urothelial neoplasia inpatients with hematuria, in patients with a history of bladdercarcinoma, or in patients at high risk of developing bladder carcinoma(screening workers exposed to aromatic amines or cadmium).

The overall sensitivity of urinary tract cytology in one large studyreported in the literature was 83 percent (reported sensitivity in theliterature varies from 47 to 97 percent). The sensitivity variesconsiderably with the grade of the neoplasm. Papillomas and grade Icarcinomas can not be reliably diagnosed. The sensitivity for grade IIcarcinomas (suspicious and positive) is 80 percent and grade III(suspicious and positive) 94 percent. Urinary tract cytology has aspecial role to play in carcinoma in situ. These tumors are oftendifficult to visualize at cystoscopy but readily shed cells. Thesensitivity for carcinoma in situ is 98 percent.

Specificity of urinary tract cytology is reported to be good with afalse positive rate of 3 percent. In addition to examination of thesediment for malignant cells, casts, renal tubular epithelial cells,dysmorphic red blood cells, and crystals may be seen in the urine ofpatients with renal parenchymal disease.

The Example: Accordingly, for this example, a sample of urine isobtained from a patient having or suspected of bladder cancer. Thesample is passed through a parylene membrane filter comprising holes ofa predetermined geometric design which is capable of capturing bladdertumor cells (e.g., shed carcinoma in situ cells). The captured cells arestained with one or more labeled antibodies capable of binding to abladder tumor antigen (e.g., UPII (uroplakin II)) which is overexpressedin bladder cancer. The stained cells are identified and counted. Thepresence or absence and or number of cells which are positivelyidentified as bladder cancer cells are used to determine the prognosisor diagnosis of the patient. The cells may be further characterized asto phenotype to assess their potential for metastasis.

Example 3 Enrichment and Capture of Circulating Endothelial Cells inBlood

In this example, circulating endothelial progenitor cells are capturedand detected.

Background to the Example: The number of circulating endothelialprogenitor cells in an individual's blood—the precursor cells to thosethat line the insides of blood vessels—may be an indicator of overallcardiovascular health. The endothelial cells lining the blood vesselsprovide essential communication between the vessels themselves andcirculating blood cells, allowing the blood to flow smoothly. Indiseases such as atherosclerosis, however, the endothelial layer becomesdamaged and the vessels do not function efficiently. Until recently,scientists believed that nearby endothelial cells were recruited to helprepair damaged blood vessels or form new ones to circumvent blockedvessels or to repair wounds. Evidence now indiicates, however, thatendothelial progenitor cells, probably generated in the bone marrow,circulate in the bloodstream and are recruited to form new blood vesselsor repair damaged ones. Endothelial cells generated in the bone marrowcontribute to continuous repair of the endothelial lining of bloodvessels. Accordingly, a lack of these cells may lead to vasculardysfunction and the progression of cardiovascular disease.

Circulating endothelial progenitor cells (CECs) are generally present inhealthy blood at a frequency of perhaps 0.5-2 cells/ml. Increasednumbers of CECs, as opposed to a decrease in endothelial progenitorcells, often up to 10-fold or more, are found in diseases and conditionsassociated with vascular perturbation or damage. Moreover, increasednumbers of endothelial cells are observed in peripheral blood of cancerpatients. These circulating endothelial progenitor cells (CECs) maycontribute to the formation of blood vessels in the tumor or reflectvascular damage caused by treatment or tumor growth.

The Example: Accordingly, in this example, a sample of blood is obtainedfrom a patient suspected of having a vascular disorder (e.g.,arteriosclerosis, a macrovascular or microvascular disorder) or cancer.The sample is passed through a parylene membrane filter comprising holesof a predetermined geometric design which is capable of capturing CECs.The captured cells are stained with one or more labeled antibodiescapable of selectively binding to the CECs. The stained cells areidentified and counted. With respect to vascular disorders, a reducedCEC count is further suggestive of a decreased capability to heal damageto the vascular endothelium. An increased count is further diagnostic ofa vascular injury or malignancy.

Example 4 Hematopoetic Stem Cells in Cord Blood

Background to the Example: Hematopoetic stem cells are primitive cellswith the ability to both multiply and separate into specific types ofcells. The body's white blood cells, red blood cells and platelets arejust a few examples of derivatives from these stem cells. Patientssuffering from a malignant disease such as leukemia may undergotreatment with radiation or chemotherapy to destroy the cancer cellsalive in their body. Radiation and chemotherapy treatments are oftensuccessful in destroying the cancer cells, however, in the process; theymay also destroy the patient's healthy cells and bone marrow.

Bone marrow is essential for the production of blood cells. If the bonemarrow is destroyed, either from a malignant, non-malignant or geneticdisorder, a stem cell transplant becomes necessary. Transplanted stemcells re-populate the bone marrow thereby replenishing the body's supplyof cells. And these hematopoetic stem cells can be found in cord blood.

The Example: Accordingly, in one aspect, cord blood is passed through aparylene membrane device which comprises a stacked series or parylenemembrane comprising a first membrane which allows red blood cells topass through and a second membrane which captures hematopoetic stemcells. A sterile physiologically acceptable rinse media which does notharm the viability of the captured cells is passed through the device.The captured stem cells are recovered from the device, optionallycultured ex vivo, and administered to a patient in need of them.

Example 5 Determination of Size Distributions of Cells or Particles

Background to the Example: Size distribution can be measured using afilter device having a plurality of parylene membrane filters inparallel. Each filter differs from others by geometrical designs whichprovide a unique critical separation threshold size. Particle or cellsize distribution in a sample can be measured by counting particlenumbers at the outlet for each filter.

Size distribution can be measured using a filter device having aplurality of parylene membrane filters stacked in a series progressingfrom larger holes to smaller holes such that each defines a retentionzone of progressively smaller particle sizes. Each filter can differsfrom others by the shape of the holes as well as cross sectional areasuch that they vary in their critical separation properties. Particle orcell distribution in a sample can be measured by counting particlesretained in each zone or retrieved from each zone. The design will besimilar to that multiple filters with different threshold hole sizeswill be stacked in series such that cells of different sizes will besorted out in different zones.

The Example: Accordingly, a sample is obtained which is suspected ofhaving particles or cells of different sizes which need to be separatedby size. The sample is passed through a parylene filter device having aplurality of parylene membrane filters stacked in a series progressingfrom larger holes to smaller holes such that each defines a retentionzone of progressively smaller particle sizes. The retention of cells atthe various membranes provides a count of the cells or particlesaccording to their size distribution.

Each publication, patent application, patent, and other reference citedherein is incorporated by reference in its entirety for all purposes tothe extent that it is not inconsistent with the present disclosure. Inparticular, all publications cited herein are incorporated herein byreference in their entirety for the purpose of describing and disclosingthe methodologies, reagents, and tools reported in the publications thatmight be used in connection with the invention. Nothing herein is to beconstrued as an admission that the invention is not entitled to antedatesuch disclosure by virtue of prior invention.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

1. A method for isolating a cancer cell from a urine sample, the methodcomprising passing the urine sample under pressure through at least afirst membrane filter consisting of a parylene substrate, a portion ofthe membrane filter having a first plurality of holes having apredetermined geometric design, wherein the first plurality of holes isbetween 5000 and 40,000 per square millimeter; a pressure generatingdevice coupled to the filter for applying pressure to a sample to befiltered; and a housing surrounding the membrane filter wherein theportion is suspended free of the housing, thereby isolating the cancercell from the urine sample.
 2. The method of claim 1, wherein the cancercell has a cross-sectional area greater than each of the first pluralityof holes and is retained by the membrane filter.
 3. The method of claim1, wherein the cancer cell is too small to be retained by the membranefilter.
 4. The method of claim 1, wherein the urine sample furthercomprises a cell of a second type and the predetermined geometric designselectively allows the cells of the second type to pass through themembrane filter while selectively limiting the passage of the cancercells.
 5. The method of claim 1, wherein the first plurality of holesare in the form of an array.
 6. The method of claim 1, wherein the firstplurality of holes provide an opening area ratio for the first membraneof from 5% to 45%.
 7. The method of claim 1, wherein the holes having apredetermined geometric design are circles, ovals, or polygons.
 8. Themethod of claim 1, wherein each of the plurality of holes has a crosssectional area ranging from about 75 square microns to about 500 squaremicrons.
 9. The method of claim 1, wherein the cancer cell is a bladdercancer cell.
 10. The method of claim 1, wherein after the fluid passesthrough the first membrane filter the cancer cell is retained on theupstream surface of the membrane filter.
 11. The method of claim 10,further comprising detection of the retained cancer cell.
 12. The methodof claim 1, wherein the sample is passed through a second membranefilter, wherein the second membrane filter comprises a second parylenemembrane having a second plurality of holes of a second predeterminedgeometric design, wherein each of the plurality of holes of the secondparylene membrane have a cross-sectional area which is smaller than thecross-sectional area of each of the holes of the first parylenemembrane, and the cancer cell passes through the first membrane filterand is retained on the surface of the second membrane filter.
 13. Themethod of claim 11, wherein the retained cancer cell is detected bycontacting the retained cancer cell with a tissue-specific or cell-typespecific antibody having a label.
 14. The method of claim 11, whereinthe retained cancer cell is contacted with a reagent capable ofdetecting a tumor associated antigen or a nucleic acid encoding thetumor associated antigen.